Establishment and Application of HRM Assay for Detection of Enzootic nasal tumor virus 2 (ENTV-2) in Goat (Capra hircus)
ZHANG Jing-Peng1, JIANG Jin-Xiu1, LIN Yu-Sheng1, YOU Wei1, LIU Dao-Quan2, MAO Kun-Min3, JIANG Bin1, HU Qi-Lin1,*
1 Institute of Animal Husbandry & Veterinary Medicine, Fujian Academy of Agricultural Sciences, Fuzhou 350013, China; 2 Fuzhou Center for Animal Disease Prevention and Control, Fuzhou 350026, China; 3 Fuqing Animal Husbandry and Veterinary Center, Fuqing 350300, China
Abstract：For the detection of enzootic nasal tumor (ENT) caused by Enzootic nasal tumor virus 2 (ENTV-2), the RT-PCR followed by restriction enzyme digestion method, RT-PCR, and qPCR had been established. However, the existence of endogenous retroviruses (ERVs) can interfere with the accuracy of the results. In this study, ENTV-2 was compared with ERVs and Jaagsiekte sheep retrovirus (JSRV) by bioinformatics methods, the conserved sequence of envelope protein (env) gene of ENTV-2 was found, and a pair of specific primers were designed. By optimizing the reaction conditions, a high resolution melting curve (HRM) detection method was established. The optimization results showed that the best reaction system was 0.4 μmol/L upstream and downstream primers, 1 μL template, 1 μL styo9 (2.5 μmol/L), 2×Premix Taq 10 μL, add ddH2O to a final volume of 20 μL. The reaction condition was 94 ℃ for 2 min; 94 ℃ for 15 s; 59 ℃ for 15 s, 72 ℃ for 15 s, 40 cycles. The env gene recombinant plasmid was constructed as standard positive plasmid and used to determine the specificity, sensitivity, and reproducibility of the HRM method. The results showed that the established HRM method could only specifically detected ENTV-2 but not ERVs which was highly homologous with ENTV-2. The sensitivity of the HRM was 82.7 copies/μL for the positive plasmid. The repeatablity of the method was good, the coefficient variations (CV) of intra-batch and inter-batch were < 1%.The positive detection rate of 92 clinical samples was 18.7%, and the coincidence rate with TaqMan detection method was 100%. This study provides technical support for the early and rapid detection of the diseases related to ENT.
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